Wound healing is a complex process where the skin or another organ or tissue repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) form a protective barrier against the external environment. If the protective barrier is broken, the normal process of wound healing is immediately set in motion. Upon injury to the skin, a set of complex biochemical events takes place to repair the damage. The classic model of wound healing is divided into several sequential, yet overlapping phases. Effective wound healing requires the highly organized integration of complex molecular and biological events including cell proliferation, migration and extracellular matrix (ECM) deposition. The speed of wound healing can be impacted by many factors, including the health of the individual and the bloodstream levels of certain hormones.
The wound healing process is not only complex but fragile, and susceptible to interruption or failure leading to the formation of non-healing chronic wounds. A non-healing wound is one that fails to heal with standard therapy in an orderly and timely manner (Troxler, M. et al., “Integrating adjunctive therapy into practice: the importance of recognizing ‘hard-to-heal’ wounds.” World Wide Wounds 2006. Available from http://www.worldwidewounds.com/2006/december/Troxler/Integrating-Adjunct-Therapy-Into-Practice.html). One of the major factors responsible for the appearance of chronic wounds is the impairment of cytokine release by local fibroblasts and inflammatory cells, which can result in reduced angiogenesis (Falange, V. (2005) “Wound healing and its impairment in the diabetic foot,” Lancet 366: 2736-1743). Many health-related factors may contribute to the development of non-healing wounds, including immunological diseases, diabetes, venous or arterial disease, advanced age, and infection.
Healing may be promoted by restoring or preventing the breakdown of the skin or tissue/organ extracellular matrix. This may be accomplished through the addition of deficient components, such as growth factors or collagen, or the introduction of a temporary matrix to support the growth of new cells or tissue. Regenerative therapies involve the use of living cells to repair, replace or restore normal function to damaged tissues and organs. Stem cells are viewed as a promising candidate for use in cell-based wound healing therapies due to their capacity for self-renewal and differentiation. Both adult and embryonic stem cells are commonly used to develop therapies for various models of disease and injury. However, a number of limitations hamper the clinical applicability of stem cells derived from adults or developing embryos, such ethical concerns and limitations on the cell sample size.
Subpopulations of stem cells exist in both the amniotic membrane and the amniotic fluid. Amniotic fluid cells are obtained during amniocentesis or scheduled C-section while amniotic membrane cells are obtained from the amnion membrane which is discarded after birth. These cells are therefore readily available, easily procured and avoid the ethical issues surrounding the use of embryonic stem cells.
Human amniotic fluid is a dynamic environment, which undergoes multiple developmental changes in order to sustain fetal growth. Fluid secretions from the fetus into the amniotic fluid carry a variety of fetal cells, resulting in a heterogeneous population of cells derived from fetal skin, gastrointestinal, respiratory and urinary tracts, and the amniotic membrane. These cells express electrolytes, growth factors, carbohydrates, lipids, proteins, amino acids, lactate, pyruvate, enzymes, hormones and other factors useful in tissue repair. Because they are readily accessible and pose little to no ethical concerns, amniotic fluid-derived cells are a promising alternative source of cells for use a strategy for cell replacement in various injury models. U.S. Patent Application Publication No. 2010/0130415 (Cohen et al.) describes formulations comprising secreted products obtained from the culture medium of stem cells, such as umbilical cord blood stem cells, for enhancement of wound healing.
Human amniotic membrane is the innermost fetal layer, lining the amniotic cavity and protecting the fetus during pregnancy. The membrane is composed of an inner amnion layer facing the fetus and a generally inelastic outer shell or chorion. Clinicians have used intact placental membrane composed of an amnion and a chorion layer in medical procedures since as early as 1910 (Davis, J. S., “Skin Transplantation with a Review of 550 Cases at the Johns Hopkins Hospital,” John Hopkins Med. J., 15:307 (1910)). As an alternative to intact placental membrane, some clinicians separate the amnion from the placental membrane, using only the amnion layer.
Certain characteristics of the placental membrane make it attractive for use by the medical community. The placental membrane has a wide number of applications in regenerative medicine, including providing scaffolding or structure for the regrowth of cells and tissue. An important advantage of placental membrane in scaffolding is that the amnion contains an epithelial layer. The epithelial cells derived from this layer are similar to stem cells, allowing the cells to differentiate into cells of the type that surrounds them. Additional cells similar to stem cells are contained in the body of the membrane, and the membrane also contains various growth and trophic factors, such as epidermal, insulin-like and fibroblast growth factors, and high concentrations of hyaluronic acid which may be beneficial in preventing scarring and inflammation and supporting healing. Thus, placental membrane offers a wide-variety of advantages for medical uses.
Although placental membranes possess many benefits and applications, availability of the membranes has limited their use. The amount of placental membrane generated from a single birth is small. As would be expected, because the supply of placental membranes is relatively small, the cost of placental membranes limits their use only to procedures that surpass a certain price or complexity. U.S. patent application Ser. Nos. 13/250,096 and 13/647,525, describe a placental membrane including a plurality of slits for increasing the membranes capacity to expand. The slits are provided through the membrane and are provided in sufficient numbers to produce a mesh-like pattern which enables the membrane to be stretched and therefore increase its length and width.
Many conditions would benefit from the application of a lattice or support system in conjunction with hormones and growth factors to treat non-healing wounds and burns. Accordingly, there is need for methods of treating wounds which utilize placental membranes in conjunction with cells derived from human amniotic fluid.